Glutamate

After being treated with dispase II for 5 min, the detached hESCs (1.0×10⁶ cells) were partially dissociated into 200–300 µm cell clusters and transferred to 6 cm bacterial-culture dishes (Alpha Plus, Taiwan) for 2 days of culturing. The differentiating medium contained DMEM-F12 (Invitrogen) and 20% knockout serum replacement (KSR, Invitrogen), 1 mM non-essential amino acids (NEAAs, Invitrogen), 2 mM glutamate (Invitrogen) and 0.1 mM 2-mercaptoethanol (Invitrogen). The day of cell dissociation was set as D1. From D3 to D4, the cell suspensions were cultured in DMEM-F12 with 1% N2 supplement (Invitrogen), 1 mM NEAAs and 2 mM glutamate. Neural inducing factors were applied during this D3–D4 stage, including 10 µM SB431542 (Sigma-Aldrich, St. Louis, MO, USA), 0.5 µM BIO (Sigma-Aldrich) and 10 ng/mL recombinant human FGF-2 (rh-FGF2, R&D Systems, Minneapolis, MN, USA). From D5, the neural inducing factors were removed, and the cells were further cultured in neurobasal media (Invitrogen) with 1% N2 supplement and 10 ng/mL rh-FGF2.

When iPSCs reach 80% confluence, cells were treated with 1 mg/ml Dispase II (Roche) and harvested by scraping (Day 0). The detached cell clumps were dissociated into 200–300 μm clusters and transferred to non-coated Petri dishes for 48 h. The differentiation medium was DMEM/F12 (Invitrogen) supplied with 20% knockout serum replacement (KSR, Invitrogen), 1 mM, non-essential amino acids (NEAA, Invitrogen), 2 mM glutamate (Invitrogen) and 100 μM 2-mercaptoethanol (Invitrogen). On Day 2 of differentiation, the cell culture medium was replaced to DMEM/F12 supplied with 1% N2 supplement (Invitrogen), 1 mM NEAAs and 2 mM glutamate. The BiSF induction factors, including 10 ng/mL recombinant human FGF-2 (rh-FGF2, R&D Systems), 10 μM SB431542 (Sigma-Aldrich) and 0.5 μM BIO (Sigma-Aldrich), were added on Day 2 for 48 h. From Day 0, 10 μM Y27632 (Cayman Chemical) was provided until the end of BiSF treatment. On Day 4, the neural induction medium was removed, and the cells were cultured in neurobasal medium (Invitrogen) with 1% N2 supplement and 10 ng/mL rh-FGF2. After neural induction, the cells were dissociated into small clumps by Accutase and seeded on 1% Geltrex (Invitrogen)-coated cell culture dishes for neural maturation. Following differentiation, NPC purity was determined by immunocytochemistry staining with Nestin antibody (Biolegend, #839801).

To assess the multiple differentiation potential, MSCs were cultured until sub-confluency and induced to differentiate into the adipogenic or osteogenic lineages for 7 and 21 days, respectively. After that, the gene expression analysis and staining were performed. The adipogenic medium consisted of α-MEM (Life Technologies), 20% FBS (Life Technologies), 2 mM glutamate (Life Technologies), 100 Units/mL penicillin/streptomycin (Sigma-Aldrich) and 55 µM 2-ME (Life Technologies), 10 mM L-ascorbic acid phosphate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 10 mg/mL insulin (Sigma-Aldrich), 0.5 mM hydrocortisone (Sigma-Aldrich), 6 mM indomethacin (R&D systems), 50 mM isobutylmethylxanthin (Enzo life science, Tokyo, Japan), as reported [66 (link)]. Osteogenic medium consisted of α-MEM (Life technologies), 20% FBS (Life Technologies), 2 mM glutamate (Life technologies), 100 Units/mL penicillin/streptomycin (Sigma-Aldrich) and 55 µM 2-ME (Life technologies), 10 mM L-ascorbic acid phosphate (FUJIFILM Wako Pure Chemical Corporation), 200 mM β-glycerophosphate (Sigma-Aldrich), as reported [67 ]. Mineralization (calcium deposition) and lipid droplet formation were evaluated, respectively, by staining with alizarin red-S or oil red-O, as reported [68 (link),69 (link)].